One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry

Abstract

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic “histone-like” peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.

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Histone H1 Post-Translational Modifications: Update and Future Perspectives

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The Impact of One Carbon Metabolism on Histone Methylation

“…In the last decade, mass spectrometry has offered a more accurate large-scale quantitative analysis of histone modifications than the traditionally used Western blot (Völker-Albert et al, 2018). A new mass spectrometry method for histone posttranslational modification (PTM) analysis provides the possibility of high throughput screening of 200 PTMs in less than a minute (Sidoli et al, 2019a). Furthermore, methods that combine metabolomics and histone PTM analysis have already been developed for acetyl-CoA and histone acetylation (Sidoli et al, 2019b).…”